A synthetic tRNA for EF‐Tu mediated selenocysteine incorporation in vivo and in vitro

Abstract

Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.

Future of the Genetic Code

“…The opening up of selenoprotein biochemistry via unconstrained proteome-wide Sec encoding enables the replacement of disulfide bridges by diselenide bridges in proteins [34,35]. As a result, metabolic engineering can be carried out replacing glutathione (GSSG) as a cellular redox buffer by selenoglutathione (GSeSeG): otherwise selenoglutathione would destabilize disulfide bridges in proteins, for the E°’ of −407 mV for GSeSeG is much more negative than that of −256 mV for GSSG [86].…”

“…The adaptation of the Sec pathway to NCAA encoding is rendered difficult by the requirement for a selenocysteine insertion sequence (SECIS) motif, and the non-binding of Sec-tRNA(Sec) to EF-Tu. However, both of these hurdles have been removed through the development of an effective tRNA(Sec) that incorporated Sec into proteins via EF-Tu binding, thus converting the highly restricted occurrence of Sec only at SECIS-directed mRNA contexts to unconstrained insertion as an NCAA anywhere in the proteome [34,35]. This liberated Sec pathway therefore can be adapted to the incorporation of NCAAs as in the case of the Pyl pathway.…”

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

“…Ambiguous decoding of Ser and Sec in this SECIS-independent route to selenoprotein synthesis supports the notion that the elaborate Sec recoding machinery may have evolved to enhance the fidelity of Sec insertion into proteins 64 . Further tRNA refinements created synthetic tRNAs 65,66 that, together with EF-Tu 67 mutants, led to efficient site-specific Sec incorporation 64 .…”

“…In E. coli , mutation of the tRNA Sec to each of the other 63 anticodons shows that Sec can be inserted in response to 60 of the codons in the genetic code 62,66 . In fact, for 15 codons, Sec-tRNA Sec can completely out-compete cognate aa-tRNA and provide selenoprotein yields that are tenfold greater than when Sec is encoded by UGA 62 .…”

Overcoming Challenges in Engineering the Genetic Code

“…Encouragingly, the E. coli tRNA identity determinants have been extensively characterized [110,130], and their antideterminants have been predicted [141], providing a starting point for the directed evolution of additional orthogonal aaRS/tRNA pairs. Additionally, preliminary work with the orthogonal selenocysteine translation machinery provides encouraging results for 60 of the 64 codons [142,143]. …”